Fig. S3.

HIRA S231A mutation affects O-GlcNAcylation of HIRA, the HIRA–UBN1 interaction, and the formation of HIRA–H3.3 complex. (A–C) Quantification of Western blot in Fig. 3E by densitometric analysis from three independent experiments. Western blot band intensities were obtained by using ImageJ. Band intensities were normalized to GFP-IP band intensity. Relative intensity is to that of WT HIRA value. Values represent mean ± SEM (*P < 0.05; **P < 0.01; n = 3 biological replicates; Student’s t test). (D) Deletion of potential UBN1 O-GlcNAcylation sites does not affect the integrity of the HIRA–H3.3 complex. GFP-tagged full-length or UBN1 deletion mutants [T861-S866 (Δ1) and T989-S1003 (Δ2)] were expressed in HEK293T cells along with Myc-tagged OGT. Total lysates were subjected to IP with GFP antibodies followed by Western blot analysis of associated proteins as indicated.